Human kinases and polynucleotides encoding the same

ABSTRACT

Novel human polynucleotide and polypeptide sequences are disclosed that can be used in therapeutic, diagnostic, and pharmacogenomic applications.

The present application is a continuation of U.S. patent applicationSer. No. 09/992,481, filed Nov. 19, 2001, now U.S. Pat. No.6,593,125issued Jul. 15, 2003, which claims the benefit of U.S. ProvisionalApplication No. 60/252,011, which was filed on Nov. 20, 2000, each ofwhich are herein incorporated by reference in their entirety.

1. INTRODUCTION

The present invention relates to the discovery, identification, andcharacterization of novel human polynucleotides encoding proteinssharing sequence similarity with animal kinases. The inventionencompasses the described polynucleotides, host cell expression systems,the encoded proteins, fusion proteins, polypeptides and peptides,antibodies to the encoded proteins and peptides, and geneticallyengineered animals that either lack or overexpress the disclosed genes,antagonists and agonists of the proteins, and other compounds thatmodulate the expression or activity of the proteins encoded by thedisclosed genes, which can be used for diagnosis, drug screening,clinical trial monitoring, the treatment of diseases and disorders, andcosmetic or nutriceutical applications.

2. BACKGROUND OF THE INVENTION

Kinases mediate the phosphorylation of a wide variety of proteins andcompounds in the cell. In conjunction with phosphatases, kinases areinvolved in a range of regulatory pathways. Given the physiologicalimportance of kinases, they have been subject to intense scrutiny andare proven drug targets.

3. SUMMARY OF THE INVENTION

The present invention relates to the discovery, identification, andcharacterization of nucleotides that encode novel human proteins and thecorresponding amino acid sequences of these proteins. The novel humanproteins (NHPs) described for the first time herein share structuralsimilarity with animal kinases, including, but not limited to, receptortyrosine kinases (SEQ ID NOS:1-2 show particular similarity to NEKfamily kinases, and SEQ ID NOS:3-5 are particularly similar to calciumand calmodulin dependent kinases as well as sequences encoding PK 80),and serine-threonine kinases. The described NHPs encode novel kinaseshaving homologues and orthologs across a range of phyla and species.

The novel human polynucleotides described herein encode open readingframes (ORFs) encoding proteins of 692 and 817 amino acids in length(see respectively SEQ ID NOS:2 and 4).

The invention also encompasses agonists and antagonists of the describedNHPs, including small molecules, large molecules, mutant NHPs, orportions thereof, that compete with native NHP, peptides, andantibodies, as well as nucleotide sequences that can be used to inhibitthe expression of the described NHPs (e.g., antisense and ribozymemolecules, and open reading frame or regulatory sequence replacementconstructs) or to enhance the expression of the described NHPs (e.g.,expression constructs that place the described polynucleotide under thecontrol of a strong promoter system), and transgenic animals thatexpress a NHP sequence, or “knock-outs” (which can be conditional) thatdo not express a functional NHP. Knock-out mice can be produced inseveral ways, one of which involves the use of mouse embryonic stemcells (“ES cells”) lines that contain gene trap mutations in a murinehomolog of at least one of the described NHPs. When the unique NHPsequences described in SEQ ID NOS:1-5 are “knocked-out” they provide amethod of identifying phenotypic expression of the particular gene aswell as a method of assigning function to previously unknown genes. Inaddition, animals in which the unique NHP sequences described in SEQ IDNOS:1-5 are “knocked-out” provide a unique source in which to elicitantibodies to homologous and orthologous proteins that would have beenpreviously viewed by the immune system as “self” and therefore wouldhave failed to elicit significant antibody responses. To these ends,gene trapped knockout ES cells have been generated in murine homologs ofthe described NHPs.

Additionally, the unique NHP sequences described in SEQ ID NOS:1-5 areuseful for the identification of protein coding sequence and mapping aunique gene to a particular chromosome (the gene encoding SEQ ID NOS:1-2is apparently encoded on human chromosome 17, see GENBANK accession no.AC010761, and the gene encoding SEQ ID NOS:3-5 is apparently encoded onhuman chromosome 3, see GENBANK accession no. AC068979). These sequencesidentify biologically verified exon splice junctions as opposed tosplice junctions that may have been bioinformatically predicted fromgenomic sequence alone. The sequences of the present invention are alsouseful as additional DNA markers for restriction fragment lengthpolymorphism (RFLP) analysis, and in forensic biology.

Further, the present invention also relates to processes for identifyingcompounds that modulate, i.e., act as agonists or antagonists, of NHPexpression and/or NHP activity that utilize purified preparations of thedescribed NHPs and/or NHP products, or cells expressing the same. Suchcompounds can be used as therapeutic agents for the treatment of any ofa wide variety of symptoms associated with biological disorders orimbalances.

4. DESCRIPTION OF THE SEQUENCE LISTING AND FIGURES

The Sequence Listing provides the sequence of the novel human ORFsencoding the described novel human kinase proteins. SEQ ID NO:5describes a NHP ORF and flanking sequences.

5. DETAILED DESCRIPTION OF THE INVENTION

The NHPs described for the first time herein are novel proteins that areexpressed in, inter alia, human cell lines and pituitary, thymus,spleen, lymph node, bone marrow, trachea, kidney, prostate, testis,thyroid, adrenal gland, pancreas, salivary gland, stomach, smallintestine, skeletal muscle, heart, uterus, placenta, adipose, skin,bladder, rectum, pericardium, ovary, fetal kidney, fetal lung, gallbladder, tongue, aorta, 6-, 9-, and 12-week embryos, adenocarcinoma,osteosarcoma, and embryonic carcinoma cells (SEQ ID NOS:1-2). SEQ IDNOS:3-5 were predominantly expressed in fetal brain, brain, spinal cord,thymus, lymph node, trachea, lung, prostate, testis, thyroid, adrenalgland, stomach, small intestine, skeletal muscle, uterus, placenta,mammary gland, skin, bladder, pericardium, hypothalamus, fetal kidney,fetal lung, tongue, aorta, 6-, 9-, and 12-week embryos, and embryoniccarcinoma cells.

The described sequences were compiled from sequences available inGENBANK, and cDNAs generated from pituitary, lymph node, mammary gland,brain, adrenal gland, fetus, and testis mRNAs (Edge Biosystems,Gaithersburg, Md.).

The present invention encompasses the nucleotides presented in theSequence Listing, host cells expressing such nucleotides, the expressionproducts of such nucleotides, and: (a) nucleotides that encode mammalianhomologs of the described genes, including the specifically describedNHPs, and the NHP products; (b) nucleotides that encode one or moreportions of an NHP that correspond to functional domains, and thepolypeptide products specified by such nucleotide sequences, including,but not limited to, the novel regions of any active domain(s); (c)isolated nucleotides that encode mutant versions, engineered ornaturally occurring, of the described NHPs in which all or a part of atleast one domain is deleted or altered, and the polypeptide productsspecified by such nucleotide sequences, including, but not limited to,soluble proteins and peptides in which all or a portion of the signalsequence is deleted; (d) nucleotides that encode chimeric fusionproteins containing all or a portion of a coding region of a NHP, or oneof its domains (e.g., a receptor/ligand binding domain, accessoryprotein/self-association domain, etc.) fused to another peptide orpolypeptide; or (e) therapeutic or diagnostic derivatives of thedescribed polynucleotides such as oligonucleotides, antisensepolynucleotides, ribozymes, dsRNA, or gene therapy constructs comprisinga sequence first disclosed in the Sequence Listing.

As discussed above, the present invention includes the human DNAsequences presented in the Sequence Listing (and vectors comprising thesame), and additionally contemplates any nucleotide sequence encoding acontiguous NHP open reading frame (ORF) that hybridizes to a complementof a DNA sequence presented in the Sequence Listing under highlystringent conditions, e.g., hybridization to filter-bound DNA in 0.5 MNaHPO₄, 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65° C., andwashing in 0.1×SSC/0.1% SDS at 68° C. (Ausubel et al., eds., 1989,Current Protocols in Molecular Biology, Vol. I, Green PublishingAssociates, Inc., and John Wiley & Sons, Inc., New York, at p. 2.10.3)and encodes a functionally equivalent expression product. Additionally,contemplated are any nucleotide sequences that hybridize to thecomplement of a DNA sequence that encodes and expresses an amino acidsequence presented in the Sequence Listing under moderately stringentconditions, e.g., washing in 0.2×SSC/0.1% SDS at 42° C. (Ausubel et al.,1989, supra), yet still encode a functionally equivalent NHP product.Functional equivalents of a NHP include naturally occurring NHPs presentin other species and mutant NHPs whether naturally occurring orengineered (by site directed mutagenesis, gene shuffling, and/ordirected evolution, as described in, for example, U.S. Pat. Nos.5,837,458 or 5,723,323, both of which are herein incorporated byreference). The invention also includes degenerate nucleic acid variantsof the disclosed NHP polynucleotide sequences.

Additionally contemplated are polynucleotides encoding NHP ORFs, ortheir functional equivalents, encoded by polynucleotide sequences thatare about 99, 95, 90, or about 85 percent similar to correspondingregions of a NHP (as measured by BLAST sequence comparison analysisusing, for example, the GCG sequence analysis package, as describedherein, using default parameters).

The invention also includes nucleic acid molecules, preferably DNAmolecules, that hybridize to, and are therefore the complements of, thedescribed NHP encoding polynucleotides. Such hybridization conditionscan be highly stringent or less highly stringent, as described herein.In instances where the nucleic acid molecules are deoxyoligonucleotides(“DNA oligos”), such molecules are generally about 16 to about 100 baseslong, or about 20 to about 80 bases long, or about 34 to about 45 baseslong, or any variation or combination of sizes represented therein thatincorporate a contiguous region of sequence first disclosed in theSequence Listing. Such oligonucleotides can be used in conjunction withthe polymerase chain reaction (PCR) to screen libraries, isolate clones,and prepare cloning and sequencing templates, etc.

Alternatively, such NHP oligonucleotides can be used as hybridizationprobes for screening libraries, and assessing gene expression patterns(particularly using a microarray or high-throughput “chip” format).Additionally, a series of the described NHP oligonucleotide sequences,or the complements thereof, can be used to represent all or a portion ofthe described NHP sequences. An oligonucleotide or polynucleotidesequence first disclosed in at least a portion of one or more of thesequences of SEQ ID NOS:1-5 can be used as a hybridization probe inconjunction with a solid support matrix/substrate (resins, beads,membranes, plastics, polymers, metal or metallized substrates,crystalline or polycrystalline substrates, etc.). Of particular note arespatially addressable arrays (i.e., gene chips, microtiter plates, etc.)of oligonucleotides and polynucleotides, or corresponding oligopeptidesand polypeptides, wherein at least one of the biopolymers present on thespatially addressable array comprises an oligonucleotide orpolynucleotide sequence first disclosed in at least one of the sequencesof SEQ ID NOS:1-5, or an amino acid sequence encoded thereby. Methodsfor attaching biopolymers to, or synthesizing biopolymers on, solidsupport matrices, and conducting binding studies thereon are disclosedin, inter alia, U.S. Pat. Nos. 5,700,637, 5,556,752, 5,744,305,4,631,211, 5,445,934, 5,252,743, 4,713,326, 5,424,186, and 4,689,405,the disclosures of which are herein incorporated by reference in theirentirety.

Addressable arrays comprising sequences first disclosed in SEQ IDNOS:1-5 can be used to identify and characterize the temporal and tissuespecific expression of a gene. These addressable arrays incorporateoligonucleotide sequences of sufficient length to confer the requiredspecificity, yet be within the limitations of the production technology.The length of these probes is within a range of between about 8 to about2000 nucleotides. Preferably the probes consist of 60 nucleotides andmore preferably 25 nucleotides from the sequences first disclosed in SEQID NOS:1-5.

For example, a series of the described oligonucleotide sequences, or thecomplements thereof, can be used in chip format to represent all or aportion of the described sequences. The oligonucleotides, typicallybetween about 16 to about 40 (or any whole number within the statedrange) nucleotides in length can partially overlap each other and/or thesequence may be represented using oligonucleotides that do not overlap.Accordingly, the described polynucleotide sequences shall typicallycomprise at least about two or three distinct oligonucleotide sequencesof at least about 8 nucleotides in length that are each first disclosedin the described Sequence Listing. Such oligonucleotide sequences canbegin at any nucleotide present within a sequence in the SequenceListing and proceed in either a sense (5′-to-3′) orientation vis-a-visthe described sequence or in an antisense orientation.

Microarray-based analysis allows the discovery of broad patterns ofgenetic activity, providing new understanding of gene functions andgenerating novel and unexpected insight into transcriptional processesand biological mechanisms. The use of addressable arrays comprisingsequences first disclosed in SEQ ID NOS:1-5 provides detailedinformation about transcriptional changes involved in a specificpathway, potentially leading to the identification of novel componentsor gene functions that manifest themselves as novel phenotypes.

Probes consisting of sequences first disclosed in SEQ ID NOS:1-5 canalso be used in the identification, selection and validation of novelmolecular targets for drug discovery. The use of these unique sequencespermits the direct confirmation of drug targets and recognition of drugdependent changes in gene expression that are modulated through pathwaysdistinct from the intended target of the drug. These unique sequencestherefore also have utility in defining and monitoring both drug actionand toxicity.

As an example of utility, the sequences first disclosed in SEQ IDNOS:1-5 can be utilized in microarrays or other assay formats, to screencollections of genetic material from patients who have a particularmedical condition. These investigations can also be carried out usingthe sequences first disclosed in SEQ ID NOS:1-5 in silico and bycomparing previously collected genetic databases and the disclosedsequences using computer software known to those in the art.

Thus the sequences first disclosed in SEQ ID NOS:1-5 can be used toidentify mutations associated with a particular disease and also indiagnostic and/or prognostic assays.

Although the presently described sequences have been specificallydescribed using nucleotide sequence, it should be appreciated that eachof the sequences can uniquely be described using any of a wide varietyof additional structural attributes, or combinations thereof. Forexample, a given sequence can be described by the net composition of thenucleotides present within a given region of the sequence in conjunctionwith the presence of one or more specific oligonucleotide sequence(s)first disclosed in the SEQ ID NOS:1-5. Alternatively, a restriction mapspecifying the relative positions of restriction endonuclease digestionsites, or various palindromic or other specific oligonucleotidesequences can be used to structurally describe a given sequence. Suchrestriction maps, which are typically generated by widely availablecomputer programs (e.g., the University of Wisconsin GCG sequenceanalysis package, SEQUENCHER 3.0, Gene Codes Corp., Ann Arbor, Mich.,etc.), can optionally be used in conjunction with one or more discretenucleotide sequence(s) present in the sequence that can be described bythe relative position of the sequence relative to one or more additionalsequence(s) or one or more restriction sites present in the disclosedsequence.

For oligonucleotide probes, highly stringent conditions may refer, e.g.,to washing in 6×SSC/0.05% sodium pyrophosphate at 37° C. (for 14-baseoligos), 48° C. (for 17-base oligos), 55° C. (for 20-base oligos), or60° C. (for 23-base oligos). These nucleic acid molecules may encode oract as NHP gene antisense molecules, useful, for example, in NHP generegulation (and/or as antisense primers in amplification reactions ofNHP gene nucleic acid sequences). With respect to NHP gene regulation,such techniques can be used to regulate biological functions. Further,such sequences can be used as part of ribozyme and/or triple helixsequences that are also useful for NHP gene regulation.

Inhibitory antisense or double stranded oligonucleotides canadditionally comprise at least one modified base moiety that is selectedfrom the group including, but not limited to, 5-fluorouracil,5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine,4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,and 2,6-diaminopurine.

The antisense oligonucleotide can also comprise at least one modifiedsugar moiety selected from the group including, but not limited to,arabinose, 2-fluoroarabinose, xylulose, and hexose.

In yet another embodiment, the antisense oligonucleotide will compriseat least one modified phosphate backbone selected from the groupincluding, but not limited to, a phosphorothioate, a phosphorodithioate,a phosphoramidothioate, a phosphoramidate, a phosphordiamidate, amethylphosphonate, an alkyl phosphotriester, and a formacetal or analogthereof.

In yet another embodiment, the antisense oligonucleotide is anα-anomeric oligonucleotide. An α-anomeric oligonucleotide forms specificdouble-stranded hybrids with complementary RNA in which, contrary to theusual β-units, the strands run parallel to each other (Gautier et al.,1987, Nucl. Acids Res. 15:6625-6641). The oligonucleotide is a2′-0-methylribonucleotide (Inoue et al., 1987, Nucl. Acids Res.15:6131-6148), or a chimeric RNA-DNA analogue (Inoue et al., 1987, FEBSLett. 215:327-330). Alternatively, double stranded RNA can be used todisrupt the expression and function of a targeted NHP.

Oligonucleotides of the invention can be synthesized by standard methodsknown in the art, e.g., by use of an automated DNA synthesizer (such asare commercially available from Biosearch, Applied Biosystems, etc.). Asexamples, phosphorothioate oligonucleotides can be synthesized (Stein etal., 1988, Nucl. Acids Res. 16:3209), and methylphosphonateoligonucleotides can be prepared by use of controlled pore glass polymersupports (Sarin et al., 1988, Proc. Natl. Acad. Sci. USA 85:7448-7451),etc.

Low stringency conditions are well-known to those of skill in the art,and will vary predictably depending on the specific organisms from whichthe library and the labeled sequences are derived. For guidanceregarding such conditions see, for example, Sambrook et al., 1989,Molecular Cloning, A Laboratory Manual (and periodic updates thereof),Cold Spring Harbor Press, N.Y.; and Ausubel et al., 1989, supra.

Alternatively, suitably labeled NHP nucleotide probes can be used toscreen a human genomic library using appropriately stringent conditionsor by PCR. The identification and characterization of human genomicclones is helpful for identifying polymorphisms (including, but notlimited to, nucleotide repeats, microsatellite alleles, singlenucleotide polymorphisms, or coding single nucleotide polymorphisms),determining the genomic structure of a given locus/allele, and designingdiagnostic tests. For example, sequences derived from regions adjacentto the intron/exon boundaries of the human gene can be used to designprimers for use in amplification assays to detect mutations within theexons, introns, splice sites (e.g., splice acceptor and/or donor sites),etc., that can be used in diagnostics and pharmacogenomics.

For example, the present sequences can be used in restriction fragmentlength polymorphism (RFLP) analysis to identify specific individuals. Inthis technique, an individuals genomic DNA is digested with one or morerestriction enzymes, and probed on a Southern blot to yield unique bandsfor identification (as generally described in U.S. Pat. No. 5,272,057,incorporated herein by reference). In addition, the sequences of thepresent invention can be used to provide polynucleotide reagents, e.g.,PCR primers, targeted to specific loci in the human genome, which canenhance the reliability of DNA-based forensic identifications by, forexample, providing another “identification marker” (i.e., another DNAsequence that is unique to a particular individual). Actual basesequence information can be used for identification as an accuratealternative to patterns formed by restriction enzyme generatedfragments.

Further, a NHP gene homolog can be isolated from nucleic acid from anorganism of interest by performing PCR using two degenerate or “wobble”oligonucleotide primer pools designed on the basis of amino acidsequences within the NHP products disclosed herein. The template for thereaction may be total RNA, mRNA, and/or cDNA obtained by reversetranscription of mRNA prepared from, for example, human or non-humancell lines or tissue known to express or suspected of expressing anallele of a NHP gene.

The PCR product can be subcloned and sequenced to ensure that theamplified sequences represent the sequence of the desired NHP gene. ThePCR fragment can then be used to isolate a full length cDNA clone by avariety of methods. For example, the amplified fragment can be labeledand used to screen a cDNA library, such as a bacteriophage cDNA library.Alternatively, the labeled fragment can be used to isolate genomicclones via the screening of a genomic library.

PCR technology can also be used to isolate full length cDNA sequences.For example, RNA can be isolated, following standard procedures, from anappropriate cellular or tissue source (i.e., one known to express, orsuspected of expressing, a NHP gene). A reverse transcription (RT)reaction can be performed on the RNA using an oligonucleotide primerspecific for the most 5′ end of the amplified fragment for the primingof first strand synthesis. The resulting RNA/DNA hybrid may then be“tailed” using a standard terminal transferase reaction, the hybrid maybe digested with RNase H, and second strand synthesis may then be primedwith a complementary primer. Thus, cDNA sequences upstream of theamplified fragment can be isolated. For a review of cloning strategiesthat can be used, see e.g., Sambrook et al., 1989, supra.

A cDNA encoding a mutant NHP sequence can be isolated, for example, byusing PCR. In this case, the first cDNA strand may be synthesized byhybridizing an oligo-dT oligonucleotide to mRNA isolated from tissueknown to express, or suspected of expressing, a mutant NHP allele, in anindividual putatively carrying a mutant NHP allele, and by extending thenew strand with reverse transcriptase. The second strand of the cDNA isthen synthesized using an oligonucleotide that hybridizes specificallyto the 5′ end of the normal sequence. Using these two primers, theproduct is then amplified via PCR, optionally cloned into a suitablevector, and subjected to DNA sequence analysis through methodswell-known to those of skill in the art. By comparing the DNA sequenceof the mutant NHP allele to that of a corresponding normal NHP allele,the mutation(s) responsible for the loss or alteration of function ofthe mutant NHP gene product can be ascertained.

Alternatively, a genomic library can be constructed using DNA obtainedfrom an individual suspected of carrying, or known to carry, a mutantNHP allele (e.g., a person manifesting a NHP-associated phenotype suchas, for example, immune disorders, obesity, high blood pressure, etc.),or a cDNA library can be constructed using RNA from a tissue known toexpress, or suspected of expressing, a mutant NHP allele. A normal NHPsequence, or any suitable fragment thereof, can then be labeled and usedas a probe to identify the corresponding mutant NHP allele in suchlibraries. Clones containing mutant NHP sequences can then be purifiedand subjected to sequence analysis according to methods well-known tothose skilled in the art.

Additionally, an expression library can be constructed utilizing cDNAsynthesized from, for example, RNA isolated from a tissue known toexpress, or suspected of expressing, a mutant NHP allele in anindividual suspected of carrying, or known to carry, such a mutantallele. In this manner, gene products made by the putatively mutanttissue may be expressed and screened using standard antibody screeningtechniques in conjunction with antibodies raised against a normal NHPproduct, as described below (for screening techniques, see, for example,Harlow and Lane, eds., 1988, “Antibodies: A Laboratory Manual”, ColdSpring Harbor Press, Cold Spring Harbor).

Additionally, screening can be accomplished by screening with labeledNHP fusion proteins, such as, for example, alkaline phosphatase-NHP orNHP-alkaline phosphatase fusion proteins. In cases where a NHP mutationresults in an expression product with altered function (e.g., as aresult of a missense or a frameshift mutation), polyclonal antibodies toa NHP are likely to cross-react with a corresponding mutant NHPexpression product. Library clones detected via their reaction with suchlabeled antibodies can be purified and subjected to sequence analysisaccording to methods well-known in the art.

An additional application of the described novel human polynucleotidesequences is their use in the molecular mutagenesis/evolution ofproteins that are at least partially encoded by the described novelsequences using, for example, polynucleotide shuffling or relatedmethodologies. Such approaches are described in U.S. Pat. Nos.5,830,721, 5,837,458, 6,117,679, and 5,723,323, which are hereinincorporated by reference in their entirety.

The invention also encompasses: (a) DNA vectors that contain any of theforegoing NHP coding sequences and/or their complements (i.e.,antisense); (b) DNA expression vectors that contain any of the foregoingNHP coding sequences operatively associated with a regulatory elementthat directs the expression of the coding sequences (for example,baculovirus as described in U.S. Pat. No. 5,869,336 herein incorporatedby reference); (c) genetically engineered host cells that contain any ofthe foregoing NHP coding sequences operatively associated with aregulatory element that directs the expression of the coding sequencesin the host cell; and (d) genetically engineered host cells that expressan endogenous NHP sequence under the control of an exogenouslyintroduced regulatory element (i.e., gene activation). As used herein,regulatory elements include, but are not limited to, inducible andnon-inducible promoters, enhancers, operators and other elements knownto those skilled in the art that drive and regulate expression. Suchregulatory elements include, but are not limited to, the cytomegalovirus(hCMV) immediate early gene, regulatable, viral elements (particularlyretroviral LTR promoters), the early or late promoters of SV40 andadenovirus, the lac system, the trp system, the TAC system, the TRCsystem, the major operator and promoter regions of phage lambda, thecontrol regions of fd coat protein, the promoter for 3-phosphoglyceratekinase (PGK), the promoters of acid phosphatase, and the promoters ofthe yeast α-mating factors.

Where, as in the present instance, some of the described NHP peptides orpolypeptides are thought to be cytoplasmic or nuclear proteins (althoughprocessed forms or fragments can be secreted or membrane associated),expression systems can be engineered that produce soluble derivatives ofa NHP (corresponding to a NHP extracellular and/or intracellulardomains, or truncated polypeptides lacking one or more hydrophobicdomains) and/or NHP fusion protein products (especially NHP-Ig fusionproteins, i.e., fusions of a NHP domain to an IgFc), NHP antibodies, andanti-idiotypic antibodies (including Fab fragments) that can be used intherapeutic applications. Preferably, the above expression systems areengineered to allow the desired peptide or polypeptide to be recoveredfrom the culture media.

The present invention also encompasses antibodies and anti-idiotypicantibodies (including Fab fragments), antagonists and agonists of a NHP,as well as compounds or nucleotide constructs that inhibit expression ofa NHP sequence (transcription factor inhibitors, antisense and ribozymemolecules, or open reading frame sequence or regulatory sequencereplacement constructs), or promote the expression of a NHP (e.g.,expression constructs in which NHP coding sequences are operativelyassociated with expression control elements such as promoters,promoter/enhancers, etc.).

The NHPs or NHP peptides, NHP fusion proteins, NHP nucleotide sequences,antibodies, antagonists and agonists can be useful for the detection ofmutant NHPs or inappropriately expressed NHPs for the diagnosis ofdisease. The NHP proteins or peptides, NHP fusion proteins, NHPnucleotide sequences, host cell expression systems, antibodies,antagonists, agonists and genetically engineered cells and animals canbe used for screening for drugs (or high throughput screening ofcombinatorial libraries) effective in the treatment of the symptomaticor phenotypic manifestations of perturbing the normal function of a NHPin the body. The use of engineered host cells and/or animals can offeran advantage in that such systems allow not only for the identificationof compounds that bind to the endogenous receptor/ligand of a NHP, butcan also identify compounds that trigger NHP-mediated activities orpathways.

Finally, the NHP products can be used as therapeutics. For example,soluble derivatives such as NHP peptides/domains corresponding to NHPs,NHP fusion protein products (especially NHP-Ig fusion proteins, i.e.,fusions of a NHP, or a domain of a NHP, to an IgFc), NHP antibodies andanti-idiotypic antibodies (including Fab fragments), antagonists oragonists (including compounds that modulate or act on downstream targetsin a NHP-mediated pathway) can be used to directly treat diseases ordisorders. For instance, the administration of an effective amount ofsoluble NHP, or a NHP-IgFc fusion protein or an anti-idiotypic antibody(or its Fab) that mimics the NHP could activate or effectivelyantagonize the endogenous NHP or a protein interactive therewith.Nucleotide constructs encoding such NHP products can be used togenetically engineer host cells to express such products in vivo; thesegenetically engineered cells function as “bioreactors” in the bodydelivering a continuous supply of a NHP, a NHP peptide, or a NHP fusionprotein to the body. Nucleotide constructs encoding functional NHPs,mutant NHPs, as well as antisense and ribozyme molecules can also beused in “gene therapy” approaches for the modulation of NHP expression.Thus, the invention also encompasses pharmaceutical formulations andmethods for treating biological disorders.

Various aspects of the invention are described in greater detail in thesubsections below.

5.1 THE NHP SEQUENCES

The cDNA sequences and corresponding deduced amino acid sequences of thedescribed NHPs are presented in the Sequence Listing.

Expression analysis has provided evidence that the described NHPs can beexpressed in a range of human tissues, as described in greater detailherein above. In addition to serine-threonine kinases, the describedNHPs also share significant similarity to several additional kinasefamilies, including kinases associated with signal transduction, from avariety of phyla and species. Several polymorphisms were identified inthe described NHPs. These include a T/C polymorphism in the sequenceregion represented by nucleotide position 1170 of SEQ ID NO:1, both ofwhich result in the same amino acid being present at the correspondingamino acid (aa) position of SEQ ID NO:2; a T/C polymorphism in thesequence region represented by nucleotide position 1321 of SEQ ID NO:1,both of which result in the same amino acid being present at thecorresponding aa position of SEQ ID NO:2; a C/G polymorphism in thesequence region represented by nucleotide position 94 of SEQ ID NO:3,which can result in either a leu or val being present at correspondingaa position 32 of SEQ ID NO:4; an A/G polymorphism at nucleotideposition 112 of SEQ ID NO:3, which can result in either a lys or glubeing present at corresponding aa position 38 of SEQ ID NO:4; and an A/Tpolymorphism at nucleotide position 133 of SEQ ID NO:3, which can resultin either a thr or ser being present at corresponding aa position 45 ofSEQ ID NO:4. The above polymorphisms can be present either singly, or inany combination or permutation within a given sequence.

An additional application of the described novel human polynucleotidesequences is their use in the molecular mutagenesis/evolution ofproteins that are at least partially encoded by the described novelsequences using, for example, polynucleotide shuffling or relatedmethodologies. Such approaches are described in U.S. Pat. Nos. 5,830,721and 5,837,458, which are herein incorporated by reference in theirentirety.

NHP gene products can also be expressed in transgenic animals. Animalsof any species, including, but not limited to, worms, mice, rats,rabbits, guinea pigs, pigs, micro-pigs, birds, goats, and non-humanprimates, e.g., baboons, monkeys, and chimpanzees may be used togenerate NHP transgenic animals.

Any technique known in the art may be used to introduce a NHP transgeneinto animals to produce the founder lines of transgenic animals. Suchtechniques include, but are not limited to, pronuclear microinjection(Hoppe and Wagner, 1989, U.S. Pat. No. 4,873,191); retrovirus mediatedgene transfer into germ lines (Van der Putten et al., 1985, Proc. Natl.Acad. Sci. USA 82:6148-6152); gene targeting in embryonic stem cells(Thompson et al., 1989, Cell 56:313-321); electroporation of embryos(Lo, 1983, Mol Cell. Biol. 3:1803-1814); and sperm-mediated genetransfer (Lavitrano et al., 1989, Cell 57:717-723); etc. For a review ofsuch techniques, see Gordon, 1989, Transgenic Animals, Intl. Rev. Cytol.115:171-229, which is incorporated by reference herein in its entirety.

The present invention provides for transgenic animals that carry the NHPtransgene in all their cells, as well as animals that carry thetransgene in some, but not all their cells, i.e., mosaic animals orsomatic cell transgenic animals. The transgene may be integrated as asingle transgene or in concatamers, e.g., head-to-head tandems orhead-to-tail tandems. The transgene may also be selectively introducedinto and activated in a particular cell type by following, for example,the teaching of Lasko et al., 1992, Proc. Natl. Acad. Sci. USA89:6232-6236. The regulatory sequences required for such a cell-typespecific activation will depend upon the particular cell type ofinterest, and will be apparent to those of skill in the art.

When it is desired that a NHP transgene be integrated into thechromosomal site of the endogenous NHP gene, gene targeting ispreferred. Briefly, when such a technique is to be utilized, vectorscontaining some nucleotide sequences homologous to the endogenous NHPgene are designed for the purpose of integrating, via homologousrecombination with chromosomal sequences, into and disrupting thefunction of the nucleotide sequence of the endogenous NHP gene (i.e.,“knockout” animals).

The transgene can also be selectively introduced into a particular celltype, thus inactivating the endogenous NHP gene in only that cell type,by following, for example, the teaching of Gu et al., 1994, Science,265:103-106. The regulatory sequences required for such a cell-typespecific inactivation will depend upon the particular cell type ofinterest, and will be apparent to those of skill in the art.

Once transgenic animals have been generated, the expression of therecombinant NHP gene may be assayed utilizing standard techniques.Initial screening may be accomplished by Southern blot analysis or PCRtechniques to analyze animal tissues to assay whether integration of thetransgene has taken place. The level of mRNA expression of the transgenein the tissues of the transgenic animals may also be assessed usingtechniques that include, but are not limited to, Northern blot analysisof tissue samples obtained from the animal, in situ hybridizationanalysis, and RT-PCR. Samples of NHP gene-expressing tissue, may also beevaluated immunocytochemically using antibodies specific for the NHPtransgene product.

5.2 NHPS AND NHP POLYPEPTIDES

NHPs, NHP polypeptides, NHP peptide fragments, mutated, truncated, ordeleted forms of the NHPs, and/or NHP fusion proteins can be preparedfor a variety of uses. These uses include, but are not limited to, thegeneration of antibodies, as reagents in diagnostic assays, for theidentification of other cellular gene products related to a NHP, asreagents in assays for screening for compounds that can be used aspharmaceutical reagents useful in the therapeutic treatment of mental,biological, or medical disorders and disease. Given the similarityinformation and expression data, the described NHPs can be targeted (bydrugs, oligos, antibodies, etc.) in order to treat disease, or totherapeutically augment the efficacy of therapeutic agents.

The Sequence Listing discloses the amino acid sequences encoded by thedescribed NHP-encoding polynucleotides. The NHPs display initiatormethionines that are present in DNA sequence contexts consistent witheucaryotic translation initiation sites. The NHPs do not displayconsensus signal sequences, which indicates that they may be cytoplasmicor possibly nuclear proteins, however, the homology data and presence ofhydrophobic domains indicates that the NHPs are probably membraneassociated, or possibly secreted.

The NHP amino acid sequences of the invention include the amino acidsequences presented in the Sequence Listing as well as analogues andderivatives thereof. Further, corresponding NHP homologues from otherspecies are encompassed by the invention. In fact, any NHP proteinencoded by the NHP nucleotide sequences described above are within thescope of the invention, as are any novel polynucleotide sequencesencoding all or any novel portion of an amino acid sequence presented inthe Sequence Listing. The degenerate nature of the genetic code iswell-known, and, accordingly, each amino acid presented in the SequenceListing, is generically representative of the well-known nucleic acid“triplet” codon, or in many cases codons, that can encode the aminoacid. As such, as contemplated herein, the amino acid sequencespresented in the Sequence Listing, when taken together with the geneticcode (see, for example, Table 4-1 at page 109 of “Molecular CellBiology”, 1986, J. Darnell et al., eds., Scientific American Books, NewYork, N.Y., herein incorporated by reference) are genericallyrepresentative of all the various permutations and combinations ofnucleic acid sequences that can encode such amino acid sequences.

The invention also encompasses proteins that are functionally equivalentto the NHPs encoded by the presently described nucleotide sequences, asjudged by any of a number of criteria, including, but not limited to,the ability to bind and modify a NHP substrate, or the ability to effectan identical or complementary downstream pathway, or a change incellular metabolism (e.g., proteolytic activity, ion flux, tyrosinephosphorylation, etc.). Such functionally equivalent NHP proteinsinclude, but are not limited to, additions or substitutions of aminoacid residues within the amino acid sequence encoded by the NHPnucleotide sequences described above, but that result in a silentchange, thus producing a functionally equivalent expression product.Amino acid substitutions may be made on the basis of similarity inpolarity, charge, solubility, hydrophobicity, hydrophilicity, and/or theamphipathic nature of the residues involved. For example, nonpolar(hydrophobic) amino acids include alanine, leucine, isoleucine, valine,proline, phenylalanine, tryptophan, and methionine; polar neutral aminoacids include glycine, serine, threonine, cysteine, tyrosine,asparagine, and glutamine; positively charged (basic) amino acidsinclude arginine, lysine, and histidine; and negatively charged (acidic)amino acids include aspartic acid and glutamic acid.

A variety of host-expression vector systems can be used to express theNHP nucleotide sequences of the invention. Where the NHP peptide orpolypeptide can exist, or has been engineered to exist, as a soluble orsecreted molecule, the soluble NHP peptide or polypeptide can berecovered from the culture media. Such expression systems also encompassengineered host cells that express a NHP, or functional equivalent, insitu. Purification or enrichment of a NHP from such expression systemscan be accomplished using appropriate detergents and lipid micelles andmethods well-known to those skilled in the art. However, such engineeredhost cells themselves may be used in situations where it is importantnot only to retain the structural and functional characteristics of theNHP, but to assess biological activity, e.g., in drug screening assays.

The expression systems that may be used for purposes of the inventioninclude, but are not limited to, microorganisms such as bacteria (e.g.,E. coli, B. subtilis) transformed with recombinant bacteriophage DNA,plasmid DNA or cosmid DNA expression vectors containing NHP nucleotidesequences; yeast (e.g., Saccharomyces, Pichia) transformed withrecombinant yeast expression vectors containing NHP nucleotidesequences; insect cell systems infected with recombinant virusexpression vectors (e.g., baculovirus) containing NHP nucleotidesequences; plant cell systems infected with recombinant virus expressionvectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus,TMV) or transformed with recombinant plasmid expression vectors (e.g.,Ti plasmid) containing NHP nucleotide sequences; or mammalian cellsystems (e.g., COS, CHO, BHK, 293, 3T3) harboring recombinant expressionconstructs containing NHP nucleotide sequences and promoters derivedfrom the genome of mammalian cells (e.g., metallothionein promoter) orfrom mammalian viruses (e.g., the adenovirus late promoter; the vacciniavirus 7.5K promoter).

In bacterial systems, a number of expression vectors may beadvantageously selected depending upon the use intended for the NHPproduct being expressed. For example, when a large quantity of such aprotein is to be produced for the generation of pharmaceuticalcompositions of or containing NHP, or for raising antibodies to a NHP,vectors that direct the expression of high levels of fusion proteinproducts that are readily purified may be desirable. Such vectorsinclude, but are not limited to, the E. coli expression vector pUR278(Ruther et al., 1983, EMBO J. 2:1791), in which a NHP coding sequencemay be ligated individually into the vector in frame with the lacZcoding region so that a fusion protein is produced; pIN vectors (Inouyeand Inouye, 1985, Nucleic Acids Res. 13:3101-3109; Van Heeke andSchuster, 1989, J. Biol. Chem. 264:5503-5509); and the like. pGEXvectors may also be used to express foreign polypeptides as fusionproteins with glutathione S-transferase (GST). In general, such fusionproteins are soluble and can easily be purified from lysed cells byadsorption to glutathione-agarose beads followed by elution in thepresence of free glutathione. The PGEX vectors are designed to includethrombin or factor Xa protease cleavage sites so that the cloned targetexpression product can be released from the GST moiety.

In an insect system, Autographa californica nuclear polyhedrosis virus(AcNPV) can be used as a vector to express foreign polynucleotidesequences. The virus grows in Spodoptera frugiperda cells. A NHP codingsequence can be cloned individually into non-essential regions (forexample the polyhedrin gene) of the virus and placed under control of anAcNPV promoter (for example the polyhedrin promoter). Successfulinsertion of NHP coding sequence will result in inactivation of thepolyhedrin gene and production of non-occluded recombinant virus (i.e.,virus lacking the proteinaceous coat coded for by the polyhedrin gene).These recombinant viruses are then used to infect Spodoptera frugiperdacells in which the inserted sequence is expressed (e.g., see Smith etal., 1983, J. Virol. 46:584; Smith, U.S. Pat. No. 4,215,051).

In mammalian host cells, a number of viral-based expression systems maybe utilized. In cases where an adenovirus is used as an expressionvector, the NHP nucleotide sequence of interest may be ligated to anadenovirus transcription/translation control complex, e.g., the latepromoter and tripartite leader sequence. This chimeric sequence may thenbe inserted in the adenovirus genome by in vitro or in vivorecombination. Insertion in a non-essential region of the viral genome(e.g., region E1 or E3) will result in a recombinant virus that isviable and capable of expressing a NHP product in infected hosts (e.g.,see Logan and Shenk, 1984, Proc. Natl. Acad. Sci. USA 81:3655-3659).Specific initiation signals may also be required for efficienttranslation of inserted NHP nucleotide sequences. These signals includethe ATG initiation codon and adjacent sequences. In cases where anentire NHP gene or cDNA, including its own initiation codon and adjacentsequences, is inserted into the appropriate expression vector, noadditional translational control signals may be needed. However, incases where only a portion of a NHP coding sequence is inserted,exogenous translational control signals, including, perhaps, the ATGinitiation codon, may be provided. Furthermore, the initiation codonshould be in phase with the reading frame of the desired coding sequenceto ensure translation of the entire insert. These exogenoustranslational control signals and initiation codons can be of a varietyof origins, both natural and synthetic. The efficiency of expression maybe enhanced by the inclusion of appropriate transcription enhancerelements, transcription terminators, etc. (see Bitter et al.; 1987,Methods in Enzymol. 153:516-544).

In addition, a host cell strain may be chosen that modulates theexpression of the inserted sequences, or modifies and processes theexpression product in the specific fashion desired. Such modifications(e.g., glycosylation) and processing (e.g., cleavage) of proteinproducts may be important for the function of the protein. Differenthost cells have characteristic and specific mechanisms for thepost-translational processing and modification of proteins andexpression products. Appropriate cell lines or host systems can bechosen to ensure the desired modification and processing of the foreignprotein expressed. To this end, eukaryotic host cells that possess thecellular machinery for proper processing of the primary transcript,glycosylation, and phosphorylation of the expression product may beused. Such mammalian host cells include, but are not limited to, CHO,VERO, BHK, HeLa, COS, MDCK, 293, 3T3, WI38, and in particular, humancell lines.

For long-term, high-yield production of recombinant proteins, stableexpression is preferred. For example, cell lines that stably express theNHP sequences described above can be engineered. Rather than usingexpression vectors that contain viral origins of replication, host cellscan be transformed with DNA controlled by appropriate expression controlelements (e.g., promoter, enhancer sequences, transcription terminators,polyadenylation sites, etc.), and a selectable marker. Following theintroduction of the foreign DNA, engineered cells may be allowed to growfor 1-2 days in an enriched media, and then are switched to a selectivemedia. The selectable marker in the recombinant plasmid confersresistance to the selection and allows cells to stably integrate theplasmid into their chromosomes and grow to form foci, which in turn canbe cloned and expanded into cell lines. This method may advantageouslybe used to engineer cell lines that express the NHP product. Suchengineered cell lines may be particularly useful in screening andevaluation of compounds that affect the endogenous activity of the NHPproduct.

A number of selection systems may be used, including, but not limitedto, the herpes simplex virus thymidine kinase (Wigler et al., 1977, Cell11:223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska andSzybalski, 1962, Proc. Natl. Acad. Sci. USA 48:2026), and adeninephosphoribosyltransferase (Lowy et al., 1980, Cell 22:817) genes, whichcan be employed in tk⁻, hgprt⁻ or aprt⁻ cells, respectively. Also,antimetabolite resistance can be used as the basis of selection for thefollowing genes: dhfr, which confers resistance to methotrexate (Wigleret al., 1980, Proc. Natl. Acad. Sci. USA 77:3567; O'Hare et al., 1981,Proc. Natl. Acad. Sci. USA 78:1527); gpt, which confers resistance tomycophenolic acid (Mulligan and Berg, 1981, Proc. Natl. Acad. Sci. USA78:2072); neo, which confers resistance to the aminoglycoside G-418(Colberre-Garapin et al., 1981, J. Mol. Biol. 150:1); and hygro, whichconfers resistance to hygromycin (Santerre et al., 1984, Gene 30:147).

Alternatively, any fusion protein can be readily purified by utilizingan antibody specific for the fusion protein being expressed. Forexample, an exemplary system allows for the ready purification ofnon-denatured fusion proteins expressed in human cell lines (Janknechtet al., 1991, Proc. Natl. Acad. Sci. USA 88:8972-8976). In this system,the sequence of interest is subcloned into a vaccinia recombinationplasmid such that the sequence's open reading frame is translationallyfused to an amino-terminal tag consisting of six histidine residues.Extracts from cells infected with recombinant vaccinia virus are loadedonto Ni²⁺-nitriloacetic acid-agarose columns and histidine-taggedproteins are selectively eluted with imidazole-containing buffers.

Also encompassed by the present invention are fusion proteins thatdirect the NHP to a target organ and/or facilitate transport across themembrane into the cytosol. Conjugation of NHPs to antibody molecules ortheir Fab fragments could be used to target cells bearing a particularepitope. Attaching the appropriate signal sequence to the NHP would alsotransport the NHP to the desired location within the cell.Alternatively, targeting of NHP or its nucleic acid sequence might beachieved using liposome or lipid complex based delivery systems. Suchtechnologies are described in “Liposomes: A Practical Approach”, New,R.R.C., ed., Oxford University Press, New York, and in U.S. Pat. Nos.4,594,595, 5,459,127, 5,948,767 and 6,110,490 and their respectivedisclosures, which are herein incorporated by reference in theirentirety. Additionally embodied are novel protein constructs engineeredin such a way that they facilitate transport of the NHP to the targetsite or desired organ, where they cross the cell membrane and/or thenucleus where the NHP can exert its functional activity. This goal maybe achieved by coupling of the NHP to a cytokine or other ligand thatprovides targeting specificity, and/or to a protein transducing domain(see generally U.S. Provisional Patent Application Ser. Nos. 60/111,701and 60/056,713, both of which are herein incorporated by reference, forexamples of such transducing sequences) to facilitate passage acrosscellular membranes, and can optionally be engineered to include nuclearlocalization.

5.3 ANTIBODIES TO NHP PRODUCTS

Antibodies that specifically recognize one or more epitopes of a NHP, orepitopes of conserved variants of a NHP, or peptide fragments of a NHP,are also encompassed by the invention. Such antibodies include, but arenot limited to, polyclonal antibodies, monoclonal antibodies (mAbs),humanized or chimeric antibodies, single chain antibodies, Fabfragments, F(ab′)₂ fragments, fragments produced by a Fab expressionlibrary, anti-idiotypic (anti-Id) antibodies, and epitope-bindingfragments of any of the above.

The antibodies of the invention can be used, for example, in thedetection of NHP in a biological sample and may, therefore, be utilizedas part of a diagnostic or prognostic technique whereby patients may betested for abnormal amounts of NHP. Such antibodies may also be utilizedin conjunction with, for example, compound screening schemes for theevaluation of the effect of test compounds on expression and/or activityof a NHP expression product. Additionally, such antibodies can be usedin conjunction gene therapy to, for example, evaluate the normal and/orengineered NHP-expressing cells prior to their introduction into thepatient. Such antibodies may additionally be used as a method for theinhibition of abnormal NHP activity. Thus, such antibodies may,therefore, be utilized as part of treatment methods.

For the production of antibodies, various host animals may be immunizedby injection with the NHP, a NHP peptide (e.g., one corresponding to afunctional domain of a NHP), truncated NHP polypeptides (NHP in whichone or more domains have been deleted), functional equivalents of theNHP, or mutated variants of the NHP. Such host animals may include, butare not limited to, pigs, rabbits, mice, goats, and rats, to name but afew. Various adjuvants may be used to increase the immunologicalresponse, depending on the host species, including, but not limited to,Freund's adjuvant (complete and incomplete), mineral salts such asaluminum hydroxide or aluminum phosphate, chitosan, surface activesubstances such as lysolecithin, pluronic polyols, polyanions, peptides,oil emulsions, and potentially useful human adjuvants such as BCG(bacille Calmette-Guerin) and Corynebacterium parvum. Alternatively, theimmune response could be enhanced by combination and or coupling withmolecules such as keyhole limpet hemocyanin, tetanus toxoid, diphtheriatoxoid, ovalbumin, cholera toxin or fragments thereof. Polyclonalantibodies are heterogeneous populations of antibody molecules derivedfrom the sera of the immunized animals.

Monoclonal antibodies, which are homogeneous populations of antibodiesto a particular antigen, can be obtained by any technique that providesfor the production of antibody molecules by continuous cell lines inculture. These include, but are not limited to, the hybridoma technique(Kohler and Milstein, 1975, Nature 256:495-497; and U.S. Pat. No.4,376,110), the human B-cell hybridoma technique (Kosbor et al., 1983,Immunology Today 4:72; Cole et al., 1983, Proc. Natl. Acad. Sci. USA80:2026-2030), and the EBV-hybridoma technique (Cole et al., 1985,Monoclonal Antibodies And Cancer Therapy, Alan R. Liss, Inc., pp.77-96). Such antibodies may be of any immunoglobulin class, includingIgG, IgM, IgE, IgA, and IgD, and any subclass thereof. The hybridomaproducing the mAbs of this invention may be cultivated in vitro or invivo. Production of high titers of mAbs in vivo makes this the presentlypreferred method of production.

In addition, techniques developed for the production of “chimericantibodies” (Morrison et al., 1984, Proc. Natl. Acad. Sci. USA,81:6851-6855; Neuberger et al., 1984, Nature, 312:604-608; Takeda etal., 1985, Nature, 314:452-454) by splicing the genes from a mouseantibody molecule of appropriate antigen specificity together with genesfrom a human antibody molecule of appropriate biological activity can beused. A chimeric antibody is a molecule in which different portions arederived from different animal species, such as those having a variableregion derived from a murine mAb and a human immunoglobulin constantregion. Such technologies are described in U.S. Pat. Nos. 6,075,181 and5,877,397 and their respective disclosures, which are hereinincorporated by reference in their entirety. Also encompassed by thepresent invention is the use of fully humanized monoclonal antibodies,as described in U.S. Pat. No. 6,150,584 and respective disclosures,which are herein incorporated by reference in their entirety.

Alternatively, techniques described for the production of single chainantibodies (U.S. Pat. No. 4,946,778; Bird, 1988, Science 242:423-426;Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; and Wardet al., 1989, Nature 341:544-546) can be adapted to produce single chainantibodies against NHP expression products. Single chain antibodies areformed by linking the heavy and light chain fragments of the Fv regionvia an amino acid bridge, resulting in a single chain polypeptide.

Antibody fragments that recognize specific epitopes may be generated byknown techniques. For example, such fragments include, but are notlimited to: F(ab′)₂ fragments, which can be produced by pepsin digestionof the antibody molecule; and Fab fragments, which can be generated byreducing the disulfide bridges of the F(ab′)₂ fragments. Alternatively,Fab expression libraries may be constructed (Huse et al., 1989, Science,246:1275-1281) to allow rapid and easy identification of monoclonal Fabfragments with the desired specificity.

Antibodies to a NHP can, in turn, be utilized to generate anti-idiotypeantibodies that “mimic” a given NHP, using techniques well-known tothose skilled in the art (see, e.g., Greenspan and Bona, 1993, FASEB J.7:437-444; and Nissinoff, 1991, J. Immunol. 147:2429-2438). For example,antibodies that bind to a NHP domain and competitively inhibit thebinding of NHP to its cognate receptor/ligand can be used to generateanti-idiotypes that “mimic” the NHP and, therefore, bind, activate, orneutralize a NHP, NHP receptor, or NHP ligand. Such anti-idiotypicantibodies, or Fab fragments of such anti-idiotypes, can be used intherapeutic regimens involving a NHP mediated pathway.

Additionally given the high degree of relatedness of mammalian NHPs, thepresently described knock-out mice (having never seen a NHP, and thusnever been tolerized to a NHP) have a unique utility, as they can beadvantageously applied to the generation of antibodies against thedisclosed mammalian NHPs (i.e., a NHP will be immunogenic in NHPknock-out animals).

The present invention is not to be limited in scope by the specificembodiments described herein, which are intended as single illustrationsof individual aspects of the invention, and functionally equivalentmethods and components are within the scope of the invention. Indeed,various modifications of the invention, in addition to those shown anddescribed herein, will become apparent to those skilled in the art fromthe foregoing description. Such modifications are intended to fallwithin the scope of the appended claims. All cited publications,patents, and patent applications are herein incorporated by reference intheir entirety.

                   #             SEQUENCE LISTING<160> NUMBER OF SEQ ID NOS: 5 <210> SEQ ID NO 1 <211> LENGTH: 2079<212> TYPE: DNA <213> ORGANISM: homo sapiens <400> SEQUENCE: 1atggagaagt acgagcggat ccgagtggtg gggagaggtg ccttcgggat tg#tgcacctg     60tgcctgcgaa aggctgacca gaagctggtg atcatcaagc agattccagt gg#aacagatg    120accaaggaag agcggcaggc agcccagaat gagtgccagg tcctcaagct gc#tcaaccac    180cccaatgtca ttgagtacta cgagaacttc ctggaagaca aagcccttat ga#tcgccatg    240gaatatgcac caggcggcac tctggctgag ttcatccaaa agcgctgtaa tt#ccctgctg    300gaggaggaga ccatcctgca cttcttcgtg cagatcctgc ttgcactgca tc#atgtgcac    360acccacctca tcctgcaccg agacctcaag acccagaaca tcctgcttga ca#aacaccgc    420atggtcgtca agatcggtga tttcggcatc tccaagatcc ttagcagcaa ga#gcaaggcc    480tacacggtgg tgggtacccc atgctatatc tcccctgagc tgtgtgaggg ca#agccctac    540aaccagaaga gtgacatctg ggccctgggc tgtgtcctct acgagctggc ca#gcctcaag    600agggctttcg aggctgcgaa cttgccagca ctggtgctga agatcatgag tg#gcaccttt    660gcacctatct ctgaccggta cagccctgag cttcgccagc tggtcctgag tc#tactcagc    720ctggagcctg cccagcggcc accactcagc cacatcatgg cacagcccct ct#gcatccgt    780gccctcctca acctccacac cgacgtgggc agtgtccgca tgcggagggc ag#agaagtcc    840gtggccccca gcaacacagg gagcaggacc accagtgtcc gctgcagagg ta#tcccccgg    900ggacctgtga ggccagccat cccaccacca ctgtcgtcag tgtatgcctg gg#gtggtggg    960ctgggcaccc ccctgcggct gccaatgctc aacacagagg tggtccaggt gg#cagctggg   1020cgcacgcaga aagccggcgt cacgcgctct gggcgtctca tcctgtggga gg#ccccaccc   1080ctaggtgcag gcggaggcag tctccttcct ggggcagtgg agcagccaca gc#cccagttc   1140atctcgcgtt tcctggaggg ccagtcgggy gtgaccatca agcacgtggc ct#gtggggac   1200ttcttcactg cctgcctgac tgacagaggc atcatcatga cattcggcag cg#gcagcaat   1260gggtgcctag gccatggcag cctcactgac atcagccagc ccaccattgt gg#aggctttg   1320ytgggctatg aaatggtgca ggtggcctgt ggggcctctc acgtgctggc cc#tgtccact   1380gagcgagaac tatttgcctg gggccgtgga gacagcggca gactggggct ag#gcaccagg   1440gagtcccaca gctgccccca gcaggtgccc atgcccccag gacaggaagc tc#agcgagtt   1500gtatgtggta tcgattcctc catgatcctc actgtgcctg gccaagccct ag#cctgtggg   1560agcaacaggt tcaacaagct gggcctggac cacctctccc tgggggagga gc#ctgtcccc   1620caccagcaag tggaggaggc cctgagcttc acactactag gctctgcacc cc#tggaccag   1680gagcctctgc tgagtataga cctgggcact gctcactcag ctgctgtgac tg#cctcgggt   1740gattgctaca cttttggcag caatcagcac ggacagttgg gcaccaatac tc#gccgaggc   1800agtcgggcac cctgtaaggt ccaaggcctt gagggcatca agatggcaat gg#tagcctgt   1860ggggatgcct tcactgtagc tattggggca gagagcgaag tgtactcttg gg#gcaaaggg   1920gcgcgaggtc gattgggaag gagggatgag gatgccggac tccctcggcc ag#tgcagttg   1980gatgagacac acccttacac ggtgacttcc gtgtcctgtt gccatggaaa ca#ccctcctg   2040 gctgttcgat cggtcacaga tgagccggtc cccccctga      #                   #  2079 <210> SEQ ID NO 2 <211> LENGTH: 692<212> TYPE: PRT <213> ORGANISM: homo sapiens <400> SEQUENCE: 2Met Glu Lys Tyr Glu Arg Ile Arg Val Val Gl #y Arg Gly Ala Phe Gly 1               5   #                10   #                15Ile Val His Leu Cys Leu Arg Lys Ala Asp Gl #n Lys Leu Val Ile Ile            20       #            25       #            30Lys Gln Ile Pro Val Glu Gln Met Thr Lys Gl #u Glu Arg Gln Ala Ala        35           #        40           #        45Gln Asn Glu Cys Gln Val Leu Lys Leu Leu As #n His Pro Asn Val Ile    50               #    55               #    60Glu Tyr Tyr Glu Asn Phe Leu Glu Asp Lys Al #a Leu Met Ile Ala Met65                   #70                   #75                   #80Glu Tyr Ala Pro Gly Gly Thr Leu Ala Glu Ph #e Ile Gln Lys Arg Cys                85   #                90   #                95Asn Ser Leu Leu Glu Glu Glu Thr Ile Leu Hi #s Phe Phe Val Gln Ile            100       #           105       #           110Leu Leu Ala Leu His His Val His Thr His Le #u Ile Leu His Arg Asp        115           #       120           #       125Leu Lys Thr Gln Asn Ile Leu Leu Asp Lys Hi #s Arg Met Val Val Lys    130               #   135               #   140Ile Gly Asp Phe Gly Ile Ser Lys Ile Leu Se #r Ser Lys Ser Lys Ala145                 1 #50                 1 #55                 1 #60Tyr Thr Val Val Gly Thr Pro Cys Tyr Ile Se #r Pro Glu Leu Cys Glu                165   #               170   #               175Gly Lys Pro Tyr Asn Gln Lys Ser Asp Ile Tr #p Ala Leu Gly Cys Val            180       #           185       #           190Leu Tyr Glu Leu Ala Ser Leu Lys Arg Ala Ph #e Glu Ala Ala Asn Leu        195           #       200           #       205Pro Ala Leu Val Leu Lys Ile Met Ser Gly Th #r Phe Ala Pro Ile Ser    210               #   215               #   220Asp Arg Tyr Ser Pro Glu Leu Arg Gln Leu Va #l Leu Ser Leu Leu Ser225                 2 #30                 2 #35                 2 #40Leu Glu Pro Ala Gln Arg Pro Pro Leu Ser Hi #s Ile Met Ala Gln Pro                245   #               250   #               255Leu Cys Ile Arg Ala Leu Leu Asn Leu His Th #r Asp Val Gly Ser Val            260       #           265       #           270Arg Met Arg Arg Ala Glu Lys Ser Val Ala Pr #o Ser Asn Thr Gly Ser        275           #       280           #       285Arg Thr Thr Ser Val Arg Cys Arg Gly Ile Pr #o Arg Gly Pro Val Arg    290               #   295               #   300Pro Ala Ile Pro Pro Pro Leu Ser Ser Val Ty #r Ala Trp Gly Gly Gly305                 3 #10                 3 #15                 3 #20Leu Gly Thr Pro Leu Arg Leu Pro Met Leu As #n Thr Glu Val Val Gln                325   #               330   #               335Val Ala Ala Gly Arg Thr Gln Lys Ala Gly Va #l Thr Arg Ser Gly Arg            340       #           345       #           350Leu Ile Leu Trp Glu Ala Pro Pro Leu Gly Al #a Gly Gly Gly Ser Leu        355           #       360           #       365Leu Pro Gly Ala Val Glu Gln Pro Gln Pro Gl #n Phe Ile Ser Arg Phe    370               #   375               #   380Leu Glu Gly Gln Ser Gly Val Thr Ile Lys Hi #s Val Ala Cys Gly Asp385                 3 #90                 3 #95                 4 #00Phe Phe Thr Ala Cys Leu Thr Asp Arg Gly Il #e Ile Met Thr Phe Gly                405   #               410   #               415Ser Gly Ser Asn Gly Cys Leu Gly His Gly Se #r Leu Thr Asp Ile Ser            420       #           425       #           430Gln Pro Thr Ile Val Glu Ala Leu Leu Gly Ty #r Glu Met Val Gln Val        435           #       440           #       445Ala Cys Gly Ala Ser His Val Leu Ala Leu Se #r Thr Glu Arg Glu Leu    450               #   455               #   460Phe Ala Trp Gly Arg Gly Asp Ser Gly Arg Le #u Gly Leu Gly Thr Arg465                 4 #70                 4 #75                 4 #80Glu Ser His Ser Cys Pro Gln Gln Val Pro Me #t Pro Pro Gly Gln Glu                485   #               490   #               495Ala Gln Arg Val Val Cys Gly Ile Asp Ser Se #r Met Ile Leu Thr Val            500       #           505       #           510Pro Gly Gln Ala Leu Ala Cys Gly Ser Asn Ar #g Phe Asn Lys Leu Gly        515           #       520           #       525Leu Asp His Leu Ser Leu Gly Glu Glu Pro Va #l Pro His Gln Gln Val    530               #   535               #   540Glu Glu Ala Leu Ser Phe Thr Leu Leu Gly Se #r Ala Pro Leu Asp Gln545                 5 #50                 5 #55                 5 #60Glu Pro Leu Leu Ser Ile Asp Leu Gly Thr Al #a His Ser Ala Ala Val                565   #               570   #               575Thr Ala Ser Gly Asp Cys Tyr Thr Phe Gly Se #r Asn Gln His Gly Gln            580       #           585       #           590Leu Gly Thr Asn Thr Arg Arg Gly Ser Arg Al #a Pro Cys Lys Val Gln        595           #       600           #       605Gly Leu Glu Gly Ile Lys Met Ala Met Val Al #a Cys Gly Asp Ala Phe    610               #   615               #   620Thr Val Ala Ile Gly Ala Glu Ser Glu Val Ty #r Ser Trp Gly Lys Gly625                 6 #30                 6 #35                 6 #40Ala Arg Gly Arg Leu Gly Arg Arg Asp Glu As #p Ala Gly Leu Pro Arg                645   #               650   #               655Pro Val Gln Leu Asp Glu Thr His Pro Tyr Th #r Val Thr Ser Val Ser            660       #           665       #           670Cys Cys His Gly Asn Thr Leu Leu Ala Val Ar #g Ser Val Thr Asp Glu        675           #       680           #       685 Pro Val Pro Pro    690 <210> SEQ ID NO 3 <211> LENGTH: 2454 <212> TYPE: DNA<213> ORGANISM: homo sapiens <400> SEQUENCE: 3atgcccgccg ccactccagc cccgcagccg ccgccgcccc cggcccggcc ag#ccccagcc     60tgcccggcgc ggcctgcccc gggacagcaa ggcctatgtg accattctct aa#aatattta    120agctcgagaa tcacagagcg gaagctgcaa ggctcctggc tgcctgccag cc#gagggaat    180ctggagaaac cattcctggg gccgcgtggc cccgtcgtgc ccttgttctg cc#ctcggaat    240ggccttcact cagcacatcc tgagaacagc cctctgaagc ccagggtcgt ga#ccgtagtg    300aagctgggtg ggcagcgccc ccgaaagatc actctgctcc tcaacaggcg at#cagtgcag    360acgttcgagc agctcttagc tgacatctca gaagccttgg gctctcccag at#ggaagaat    420gaccgtgtga ggaaactgtt taacctcaag ggcagggaaa tcaggagcgt ct#ctgatttc    480ttcagggaag gggatgcttt catagctatg ggcaaagaac cactgacact ga#agagcatt    540caggtggctg tagaagaact gtaccccaac aaagcccggg ccctgacact gg#cccagcac    600agccgtgccc cttctccaag gctgaggagc aggctgttta gcaaggctct ga#aaggagac    660caccgctgtg gggagaccga gacccccaag agctgcagcg aagttgcagg at#gcaaggca    720gccatgaggc accaggggaa gatccccgag gagctttcac tagatgacag ag#cgaggacc    780cagaagaagt gggggagggg gaaatgggag ccagaaccca gtagcaagcc cc#ccagggaa    840gccactctgg aagagaggca cgcaagggga gagaagcatc ttggggtgga ga#ttgaaaag    900acctcgggtg aaattatcag atgcgagaag tgcaagagag agagggagct tc#agcagagc    960ctggagcgtg agaggctttc tctggggacc agtgagctgg atatggggaa gg#gcccaatg   1020tatgatgtgg agaagctggt gaggaccaga agctgcagga ggtctcccga gg#caaatcct   1080gcaagtgggg aggaagggtg gaagggtgac agccacagga gcagccccag ga#atcccact   1140caagagctga ggagacccag caagagcatg gacaagaaag aggacagagg cc#cagaggat   1200caagaaagcc atgctcaggg agcagccaag gccaagaagg accttgtgga ag#ttcttcct   1260gtcacagagg aggggctgag ggaggtgaag aaggacacca ggcccatgag ca#ggagcaaa   1320catggtggct ggctcctgag agagcaccag gcgggctttg agaagctccg ca#ggacccga   1380ggagaagaga aggaggcaga gaaggagaaa aagccatgta tgtctggagg ca#gaaggatg   1440actctcagag atgaccaacc tgcaaagcta gaaaaggagc ccaagacgag gc#cagaagag   1500aacaagccag agcggcccag cggtcggaag ccacggccca tgggcatcat tg#ccgccaat   1560gtggaaaagc attatgagac tggccgggtc attggggatg ggaactttgc tg#tcgtgaag   1620gagtgcagac accgcgagac caggcaggcc tatgcgatga agatcattga ca#agtccaga   1680ctcaagggca aggaggacat ggtggacagt gagatcttga tcatccagag cc#tctctcac   1740cccaacatcg tgaaattgca tgaagtctac gaaacagaca tggaaatcta cc#tgatcctg   1800gagtacgtgc agggaggaga cctttttgac gccatcatag aaagtgtgaa gt#tcccggag   1860cccgatgctg ccctcatgat catggactta tgcaaagccc tcgtccacat gc#acgacaag   1920agcattgtcc accgggacct caagccggaa aaccttttgg ttcagcgaaa tg#aggacaaa   1980tctactacct tgaaattggc tgattttgga cttgcaaagc atgtggtgag ac#ctatattt   2040actgtgtgtg ggaccccaac ttacgtagct cccgaaattc tttctgagaa ag#gttatgga   2100ctggaggtgg acatgtgggc tgctggcgtg atcctctata tcctgctgtg tg#gctttccc   2160ccattccgca gccctgagag ggaccaggac gagctcttta acatcatcca gc#tgggccac   2220tttgagttcc tcccccctta ctgggacaat atctctgatg ctgctaaaga tc#tggtgagc   2280cggttgctgg tggtagaccc caaaaagcgc tacacagctc atcaggttct tc#agcacccc   2340tggatcgaaa cagctggcaa gaccaataca gtgaaacgac agaagcaggt gt#cccccagc   2400agcgatggtc acttccggag ccagcacaag agggttgtgg agcaggtatc at#ag         2454 <210> SEQ ID NO 4 <211> LENGTH: 817 <212> TYPE: PRT<213> ORGANISM: homo sapiens <400> SEQUENCE: 4Met Pro Ala Ala Thr Pro Ala Pro Gln Pro Pr #o Pro Pro Pro Ala Arg 1               5   #                10   #                15Pro Ala Pro Ala Cys Pro Ala Arg Pro Ala Pr #o Gly Gln Gln Gly Leu            20       #            25       #            30Cys Asp His Ser Leu Lys Tyr Leu Ser Ser Ar #g Ile Thr Glu Arg Lys        35           #        40           #        45Leu Gln Gly Ser Trp Leu Pro Ala Ser Arg Gl #y Asn Leu Glu Lys Pro    50               #    55               #    60Phe Leu Gly Pro Arg Gly Pro Val Val Pro Le #u Phe Cys Pro Arg Asn65                   #70                   #75                   #80Gly Leu His Ser Ala His Pro Glu Asn Ser Pr #o Leu Lys Pro Arg Val                85   #                90   #                95Val Thr Val Val Lys Leu Gly Gly Gln Arg Pr #o Arg Lys Ile Thr Leu            100       #           105       #           110Leu Leu Asn Arg Arg Ser Val Gln Thr Phe Gl #u Gln Leu Leu Ala Asp        115           #       120           #       125Ile Ser Glu Ala Leu Gly Ser Pro Arg Trp Ly #s Asn Asp Arg Val Arg    130               #   135               #   140Lys Leu Phe Asn Leu Lys Gly Arg Glu Ile Ar #g Ser Val Ser Asp Phe145                 1 #50                 1 #55                 1 #60Phe Arg Glu Gly Asp Ala Phe Ile Ala Met Gl #y Lys Glu Pro Leu Thr                165   #               170   #               175Leu Lys Ser Ile Gln Val Ala Val Glu Glu Le #u Tyr Pro Asn Lys Ala            180       #           185       #           190Arg Ala Leu Thr Leu Ala Gln His Ser Arg Al #a Pro Ser Pro Arg Leu        195           #       200           #       205Arg Ser Arg Leu Phe Ser Lys Ala Leu Lys Gl #y Asp His Arg Cys Gly    210               #   215               #   220Glu Thr Glu Thr Pro Lys Ser Cys Ser Glu Va #l Ala Gly Cys Lys Ala225                 2 #30                 2 #35                 2 #40Ala Met Arg His Gln Gly Lys Ile Pro Glu Gl #u Leu Ser Leu Asp Asp                245   #               250   #               255Arg Ala Arg Thr Gln Lys Lys Trp Gly Arg Gl #y Lys Trp Glu Pro Glu            260       #           265       #           270Pro Ser Ser Lys Pro Pro Arg Glu Ala Thr Le #u Glu Glu Arg His Ala        275           #       280           #       285Arg Gly Glu Lys His Leu Gly Val Glu Ile Gl #u Lys Thr Ser Gly Glu    290               #   295               #   300Ile Ile Arg Cys Glu Lys Cys Lys Arg Glu Ar #g Glu Leu Gln Gln Ser305                 3 #10                 3 #15                 3 #20Leu Glu Arg Glu Arg Leu Ser Leu Gly Thr Se #r Glu Leu Asp Met Gly                325   #               330   #               335Lys Gly Pro Met Tyr Asp Val Glu Lys Leu Va #l Arg Thr Arg Ser Cys            340       #           345       #           350Arg Arg Ser Pro Glu Ala Asn Pro Ala Ser Gl #y Glu Glu Gly Trp Lys        355           #       360           #       365Gly Asp Ser His Arg Ser Ser Pro Arg Asn Pr #o Thr Gln Glu Leu Arg    370               #   375               #   380Arg Pro Ser Lys Ser Met Asp Lys Lys Glu As #p Arg Gly Pro Glu Asp385                 3 #90                 3 #95                 4 #00Gln Glu Ser His Ala Gln Gly Ala Ala Lys Al #a Lys Lys Asp Leu Val                405   #               410   #               415Glu Val Leu Pro Val Thr Glu Glu Gly Leu Ar #g Glu Val Lys Lys Asp            420       #           425       #           430Thr Arg Pro Met Ser Arg Ser Lys His Gly Gl #y Trp Leu Leu Arg Glu        435           #       440           #       445His Gln Ala Gly Phe Glu Lys Leu Arg Arg Th #r Arg Gly Glu Glu Lys    450               #   455               #   460Glu Ala Glu Lys Glu Lys Lys Pro Cys Met Se #r Gly Gly Arg Arg Met465                 4 #70                 4 #75                 4 #80Thr Leu Arg Asp Asp Gln Pro Ala Lys Leu Gl #u Lys Glu Pro Lys Thr                485   #               490   #               495Arg Pro Glu Glu Asn Lys Pro Glu Arg Pro Se #r Gly Arg Lys Pro Arg            500       #           505       #           510Pro Met Gly Ile Ile Ala Ala Asn Val Glu Ly #s His Tyr Glu Thr Gly        515           #       520           #       525Arg Val Ile Gly Asp Gly Asn Phe Ala Val Va #l Lys Glu Cys Arg His    530               #   535               #   540Arg Glu Thr Arg Gln Ala Tyr Ala Met Lys Il #e Ile Asp Lys Ser Arg545                 5 #50                 5 #55                 5 #60Leu Lys Gly Lys Glu Asp Met Val Asp Ser Gl #u Ile Leu Ile Ile Gln                565   #               570   #               575Ser Leu Ser His Pro Asn Ile Val Lys Leu Hi #s Glu Val Tyr Glu Thr            580       #           585       #           590Asp Met Glu Ile Tyr Leu Ile Leu Glu Tyr Va #l Gln Gly Gly Asp Leu        595           #       600           #       605Phe Asp Ala Ile Ile Glu Ser Val Lys Phe Pr #o Glu Pro Asp Ala Ala    610               #   615               #   620Leu Met Ile Met Asp Leu Cys Lys Ala Leu Va #l His Met His Asp Lys625                 6 #30                 6 #35                 6 #40Ser Ile Val His Arg Asp Leu Lys Pro Glu As #n Leu Leu Val Gln Arg                645   #               650   #               655Asn Glu Asp Lys Ser Thr Thr Leu Lys Leu Al #a Asp Phe Gly Leu Ala            660       #           665       #           670Lys His Val Val Arg Pro Ile Phe Thr Val Cy #s Gly Thr Pro Thr Tyr        675           #       680           #       685Val Ala Pro Glu Ile Leu Ser Glu Lys Gly Ty #r Gly Leu Glu Val Asp    690               #   695               #   700Met Trp Ala Ala Gly Val Ile Leu Tyr Ile Le #u Leu Cys Gly Phe Pro705                 7 #10                 7 #15                 7 #20Pro Phe Arg Ser Pro Glu Arg Asp Gln Asp Gl #u Leu Phe Asn Ile Ile                725   #               730   #               735Gln Leu Gly His Phe Glu Phe Leu Pro Pro Ty #r Trp Asp Asn Ile Ser            740       #           745       #           750Asp Ala Ala Lys Asp Leu Val Ser Arg Leu Le #u Val Val Asp Pro Lys        755           #       760           #       765Lys Arg Tyr Thr Ala His Gln Val Leu Gln Hi #s Pro Trp Ile Glu Thr    770               #   775               #   780Ala Gly Lys Thr Asn Thr Val Lys Arg Gln Ly #s Gln Val Ser Pro Ser785                 7 #90                 7 #95                 8 #00Ser Asp Gly His Phe Arg Ser Gln His Lys Ar #g Val Val Glu Gln Val                805   #               810   #               815 Ser<210> SEQ ID NO 5 <211> LENGTH: 2824 <212> TYPE: DNA<213> ORGANISM: homo sapiens <400> SEQUENCE: 5cgggctcgtg gctgctcgtc tcgccccgcc ttcccgcgcc tgctcgaccg tc#gagccgcg     60tccccgcgct gccacctctg ctccaggctc tccccgagcc cgccgccgcg cc#atgcccgc    120cgccactcca gccccgcagc cgccgccgcc cccggcccgg ccagccccag cc#tgcccggc    180gcggcctgcc ccgggacagc aaggcctatg tgaccattct ctaaaatatt ta#agctcgag    240aatcacagag cggaagctgc aaggctcctg gctgcctgcc agccgaggga at#ctggagaa    300accattcctg gggccgcgtg gccccgtcgt gcccttgttc tgccctcgga at#ggccttca    360ctcagcacat cctgagaaca gccctctgaa gcccagggtc gtgaccgtag tg#aagctggg    420tgggcagcgc ccccgaaaga tcactctgct cctcaacagg cgatcagtgc ag#acgttcga    480gcagctctta gctgacatct cagaagcctt gggctctccc agatggaaga at#gaccgtgt    540gaggaaactg tttaacctca agggcaggga aatcaggagc gtctctgatt tc#ttcaggga    600aggggatgct ttcatagcta tgggcaaaga accactgaca ctgaagagca tt#caggtggc    660tgtagaagaa ctgtacccca acaaagcccg ggccctgaca ctggcccagc ac#agccgtgc    720cccttctcca aggctgagga gcaggctgtt tagcaaggct ctgaaaggag ac#caccgctg    780tggggagacc gagaccccca agagctgcag cgaagttgca ggatgcaagg ca#gccatgag    840gcaccagggg aagatccccg aggagctttc actagatgac agagcgagga cc#cagaagaa    900gtgggggagg gggaaatggg agccagaacc cagtagcaag ccccccaggg aa#gccactct    960ggaagagagg cacgcaaggg gagagaagca tcttggggtg gagattgaaa ag#acctcggg   1020tgaaattatc agatgcgaga agtgcaagag agagagggag cttcagcaga gc#ctggagcg   1080tgagaggctt tctctgggga ccagtgagct ggatatgggg aagggcccaa tg#tatgatgt   1140ggagaagctg gtgaggacca gaagctgcag gaggtctccc gaggcaaatc ct#gcaagtgg   1200ggaggaaggg tggaagggtg acagccacag gagcagcccc aggaatccca ct#caagagct   1260gaggagaccc agcaagagca tggacaagaa agaggacaga ggcccagagg at#caagaaag   1320ccatgctcag ggagcagcca aggccaagaa ggaccttgtg gaagttcttc ct#gtcacaga   1380ggaggggctg agggaggtga agaaggacac caggcccatg agcaggagca aa#catggtgg   1440ctggctcctg agagagcacc aggcgggctt tgagaagctc cgcaggaccc ga#ggagaaga   1500gaaggaggca gagaaggaga aaaagccatg tatgtctgga ggcagaagga tg#actctcag   1560agatgaccaa cctgcaaagc tagaaaagga gcccaagacg aggccagaag ag#aacaagcc   1620agagcggccc agcggtcgga agccacggcc catgggcatc attgccgcca at#gtggaaaa   1680gcattatgag actggccggg tcattgggga tgggaacttt gctgtcgtga ag#gagtgcag   1740acaccgcgag accaggcagg cctatgcgat gaagatcatt gacaagtcca ga#ctcaaggg   1800caaggaggac atggtggaca gtgagatctt gatcatccag agcctctctc ac#cccaacat   1860cgtgaaattg catgaagtct acgaaacaga catggaaatc tacctgatcc tg#gagtacgt   1920gcagggagga gacctttttg acgccatcat agaaagtgtg aagttcccgg ag#cccgatgc   1980tgccctcatg atcatggact tatgcaaagc cctcgtccac atgcacgaca ag#agcattgt   2040ccaccgggac ctcaagccgg aaaacctttt ggttcagcga aatgaggaca aa#tctactac   2100cttgaaattg gctgattttg gacttgcaaa gcatgtggtg agacctatat tt#actgtgtg   2160tgggacccca acttacgtag ctcccgaaat tctttctgag aaaggttatg ga#ctggaggt   2220ggacatgtgg gctgctggcg tgatcctcta tatcctgctg tgtggctttc cc#ccattccg   2280cagccctgag agggaccagg acgagctctt taacatcatc cagctgggcc ac#tttgagtt   2340cctcccccct tactgggaca atatctctga tgctgctaaa gatctggtga gc#cggttgct   2400ggtggtagac cccaaaaagc gctacacagc tcatcaggtt cttcagcacc cc#tggatcga   2460aacagctggc aagaccaata cagtgaaacg acagaagcag gtgtccccca gc#agcgatgg   2520tcacttccgg agccagcaca agagggttgt ggagcaggta tcatagtcac ca#ccttggga   2580atctgtccag cccccagttc tgctcaagga cagagaaaag gatagaagtt tg#agagaaaa   2640acaatgaaag aggcttcttc acataattgg tgaatcagag ggagagacac tg#agtatatt   2700ttaaagcata ttaaaaaaat taagtcaatg ttaaatgtca caacatattt tt#agatttgt   2760atatttaaag cctttaatac atttttgggg ggtaagcatt gtcatcagtg ag#gaattttg   2820 gtaa                  #                  #                   #           2824

What is claimed is:
 1. An isolated polypeptide comprising the amino acidsequence of SEQ ID NO:2 or SEQ ID NO:4.
 2. A substantially isolatedprotein having the kinase activity of the protein shown in SEQ ID NO:2,which is encoded by a nucleotide sequence that hybridizes to SEQ ID NO:1under highly stringent conditions.
 3. The substantially isolated proteinof claim 2, comprising the amino acid sequence of SEQ ID NO:2.
 4. Asubstantially isolated protein having the kinase activity of the proteinshown in SEQ ID NO:4, which is encoded by a nucleotide sequence thathybridizes to SEQ ID NO:3 under highly stringent conditions.
 5. Thesubstantially isolated protein of claim 4, comprising the amino acidsequence of SEQ ID NO:4.